The world is rapidly heading towards a pre-1940's scenario when it comes to fighting infectious disease. Antimicrobial resistance is a growing problem on a global scale, greatly hampering our abilities to quell worldwide epidemics such as tuberculosis and malaria, as well as the simple staphylococcus infection. The proposed project is significant and has huge potential for impact on public health because unless innovative strategies are developed to produce robust and effective new classes of antibiotics, health care costs will continue to climb and we will completely lose our ability to combat even the most common infection. Current antibiotic treatments originated predominantly from natural products produced by fungi and bacteria that were able to inhibit the growth of other organisms, usually by inhibiting cell wall synthesis or maintenance or by inhibiting protein synthesis. Since penicilln was first isolated by Fleming in 1929, most of the subsequent generations of antibiotics remain very similar to the original natural products, with functional groups modified to increase their activity across a broader range of pathogens and decrease their side effect profiles. Oxazolidones, glycopeptides, -lactams, and quinolones show some promise for the future, but gram-negative bacterial infections still remain problematic. Nucleic acids are promising avenues for drug design, both as therapeutics and as targets. However, specificity is often a problem with small molecule nucleic acid binders such as intercalators, groove binders, and even aminoglycosides. Here we propose an innovative plan for identification of, and both functional and mechanistic assaying of, a novel class of aminoglycoside-nucleic acid conjugate ligands that are specific for an aminoglycoside-targeting riboswitch and render it inactive in vivo. This riboswitch is a key switch in the mechanism responsible for conferring antibiotic resistance in dozens of pathogenic bacterial strains, and has never before been targeted for possible therapeutic development, to our knowledge. The designed ligands, which are aminoglycoside conjugates, have the potential to be both specific for this riboswitch target, and useful against a broad spectrum of infectious bacteria, including gram- negative strains. First, as outlined in Specific Aim 1, we will obtain a model riboswitch aptamer domain that has been synthesized commercially with FRET donor and acceptor dyes in different regions of the construct. We will perform a fluorescence assay to rapidly screen approximately 80 novel aminoglycoside-nucleic acid conjugates developed at NUBAD LLC for binding to the riboswitch target, and identify promising ligands with high specificity and affinity for the target riboswitch (as outlined in Specific Aim 2). In vivo assays will be used (Specific Aim 2) to identify lead compounds that are uptaken by aminoglycoside resistant cells and render them susceptible aminoglycosides once again. In order to verify that the compounds indeed inhibit the riboswitch's mechanism of action, mechanistic assays will be performed (Specific Aim 3). The riboswitch will be positioned within a reporter plasmid so that it is under control of an IPTG-inducible tac promoter (Ptac) that will be positioned upstream of the ?-gal reporter gene. Function of the riboswitch will be assessed by agar diffusion analysis in the presence of aminoglycosides and selected identified conjugate ligand binders. As a result of this study, several lead compounds will be identified that (1) are taken up by pathogenic bacteria; (2) restore aminoglycoside susceptibility to resistant bacteria, and (3) specifically target the aminoglycoside-binding riboswitch as their primary mechanism of action. Future phases of this project will focus on developing these lead compounds for development as therapeutics. NUBAD LLC is a drug discovery company devoted to identifying therapeutic agents that target nucleic acids. We develop novel probes, assays and small molecule therapeutics targeting RNA and DNA structures identified as targets in human disease, and this project is extremely well-suited to NUBAD's aims and its employees' specific skill sets.